Rare cell detection using flat-panel imager and chemiluminescent or radioisotopic tags

ABSTRACT

Disclosed is a method using a large area flat panel imager which is specifically adapted for rare cell detection methods. The method generally includes an imager having a sample receiving surface which can provide a digital or electronic image of a sample deposited on the surface. The method also includes a selectively positionable microscope and/or camera which are used to obtain high resolution images of the deposited samples. An electronic controller can also be used in conjunction with the imager, microscope, and/or camera to selectively position at least one of those components to focus on desired regions of the deposited sample. The noted method is particularly adapted for use with chemiluminescence or other tagging technologies.

INCORPORATION BY REFERENCE

This is a divisional of application of U.S. Ser. No. 10/937,009, filedSep. 9, 2004, entitled “Rare Cell Detection Using Flat-Panel Imager AndChemiluminescent Or Radioisotopic Tags”, by Huangpin Ben Hsieh, et al.,the disclosure of which is hereby incorporated by reference in itsentirety.

BACKGROUND

The present exemplary embodiment relates to cell screening techniques.It finds particular application in conjunction with cell screeningmethods and related instrumentation, and will be described withparticular reference thereto. However, it is to be appreciated that thepresent exemplary embodiment is also amenable to other likeapplications.

Many medical applications would benefit from the ability to detect rarecell events. For example, it is known that fetal cells circulate in thematernal blood stream. See Oosterwijk et.al., Am. J. Hum. Genet., 63:1783-1792 (1998); Bajaj et. al., Cytometry, 39: 285-294 (2000), thecontents of which are incorporated by reference herein. Cancer cells canbe found circulating in the bloodstream depending on the stage ofcancer. See Hochten-Wollmar et. al., Int. J. Cancer, 70: 396-400 (1997),the contents of which are incorporated by reference herein. Shed tissuesof people infected with viruses can also be detected in the circulationsystem at an early stage. See Musiani et. al., J. Histochem. Cytochem.,45(5): 729-735 (1997), the contents of which are incorporated byreference herein. Early detection of these rare cells would allowinvasive diagnostic procedures such as amniocentesis to be avoided,cancer development to be properly monitored and treatment to beprescribed, or viral outbreak to be prevented. Detection of these rarecells would also find use in other diagnostic or research applications.

To provide statistically significant information, it is necessary toscreen about 50-100 million blood cells in order to detect rare cellevents occurring at scales of 1 in 1 to 10 millions. Therefore, a systemthat could efficiently and quickly process a large number of cells, suchas up to 100 million, at a time would be beneficial. However, thesenumbers are exemplary and should not be construed as limiting.

Measuring emitted light has become a widely used method in detectingrare cell events. Two methods may be used to create these emittedphotons. Chemiluminescence (CL) refers to the light emitted by achemical reaction, especially when the chemical reaction is catalyzed byan enzyme. For example, luciferase catalyzes the oxidation of luciferinand produces green light. Alkaline phosphatase can be used with1,2-dioxetane substrates such as CSPD and CDP-Star to produce light aswell. In one method, the enzyme is linked to an antibody specific to aprotein marker and the antibody binds the protein. Reagents areintroduced which are cleaved by the enzyme and cause CL. A secondanalytical technique known as fluorescence uses a molecule (usually anorganic dye or fluorchrome but more recently inorganic semiconductornanocrystal material such as quantum dot is also used as a taggingmolecule) which absorbs light at one wavelength and emits light at adifferent wavelength. The molecule is first bound to a cell or cellcomponent such as a protein, lipid, chromosome, or other suchcomponents. A light (usually a laser) is then used at the wavelengthabsorbed by the molecule to excite it and the amount of light emitted atthe emission wavelength is measured. These assays are useful because theamount of light emitted is proportional to, and thus can be used todetermine the number of cells or cell components to which the moleculeis bound that is present in the sample. Similarly, the rate of lightoutput can also be used to determine the concentration of the cells orcell components present in the sample. These assays are very sensitive,have a wide dynamic range within which they can be used, and have lowbackground noise. The kinetics of CL also allow for two different typesof kinetics. In “flash” kinetics, light output peaks rapidly, then diesoff quickly. In “glow” kinetics, light output is steady for acomparatively long period of time. Because of these characteristics,they are widely used in studying gene expression and regulation withinliving cells as well as for protein/nucleic acid blots.

Cell detection based on illumination to generate fluorescence iscurrently used in two main categories: flow cytometry and, imagecytometry, which includes conventional or digital microscopy as well aslaser scanning microscopy. In flow cytometry, cells are suspended insolution and travel one by one past a sensing point. Fluorescentcompounds can be attached to the cells or cell components and detectedusing a laser which excites the compound and causes it to fluoresce. Inconventional fluorescent microscopy, cells are fixed on a slide andstained with a fluorescent compound, then viewed under a microscope.Broad spectrum light sources (such as mercury arc lamp or Xenon flashlamp) coupled with specific excitation/emission filters are used toblock undesired excitation lights and allow detection of weakerfluorescence. They usually include automatic stage movement and uselow-magnification scanning to identify potential cells of interestfollowed by high magnification to reject false positives. Bajajdescribed one such setup of a fluorescent microscope. It's also notedthat laser can be used to excite these fluorescent compounds in a systemcalled laser scanning cytometer (LSC). Both of these image-basedcytometry methods direct the excitation light and collect thefluorescence through a microscope objective.

However, both flow cytometry and fluorescent microscopy have severalproblems which hinder their use in clinical applications. Bajaj et al.concluded that flow cytometry could be used to screen cells quickly, butgenerated high numbers of false positives due to autofluorescence,nonspecific staining, and cell aggregates. See also Radbruch et. al.,Curr. Opin. lmmunol. 7:270-273 (1995), the contents of which areincorporated by reference herein. Also, in practice a high number ofcells bunch or clump together, making it impossible to examine each cellseparately. The cells being examined cannot be saved for confirmation ofthe diagnosis, nor can the high-resolution images needed for suchconfirmation be taken by current instrumentation.

Conventional fluorescent microscopy or laser scanning cytometry areunsuitable for clinical use because it usually requires at least 10-20microscope slides and from 5 to 30 hours to scan 100 million bloodcells. In addition, the use of fluorescence creates the need to filterstrong exciting light and also leads to the possibility of bleaching,both of which affect the sensitivity of the assay. However, microscopyallows the cells to be saved for confirmation or for images to becaptured either digitally or on film. Finally, the equipment costs ofboth techniques are very high.

A significant cause of delay in imaging is related to the process bywhich cells or samples are examined. In current image cytometry systems,the cell samples are usually processed in two steps. First, the imagingdevice (e.g., a microscope with a CCD camera) is used at low resolutionand passes over the entire area of the slide in order to detect all“potential hits.” However, with a typical field of view of only a fewsquare millimeters through a low-magnification 4× objective and a CCDcamera, moving the objective across entire surfaces of multiple slidesis a very time-consuming bottleneck. After this “pre-screening,” theimaging device then reexamines the “potential hits” at high resolution(such as 20× or 40×) by making a second pass over the slide. This secondpass increases the amount of time required for a complete examination ofthe cell samples.

There is therefore a need for methods and systems which are lessexpensive, have higher throughput of cells, and allow for confirmationof results. The present exemplary embodiment contemplates a new andimproved approach for rare cell detection methods and systems whichovercome the above-referenced problems and others.

BRIEF DESCRIPTION

In accordance with another aspect of the present exemplary embodiment, amethod is provided for detecting the presence of tagged biologicalagents in a sample. The method comprises a step of providing a systemhaving an imager, a microscope, a camera, and a sample receivingsurface. The method also comprises a step of providing a samplecontaining a biological agent to be detected. The method comprises astep of tagging the biological agent to be detected within the sample.The method also comprises a step of depositing the sample upon thesample receiving surface. The method further comprises a step ofobtaining a digital image of the deposited tagged sample from thesystem. The method additionally comprises a step of analyzing thedigital image and selecting one or more regions of the tagged sample forhigh resolution examination. The method also comprises a step ofpositioning the imager and microscope-camera relative to each other sothe microscope can image a selected region of the tagged sampledetermined in the analyzing step. The method also comprises a step ofobtaining a second digital image of the selected region of the taggedsample. And, the method comprises a step of confirming the detection ofthe tagged biological agent. The method also comprises a step to ensurea darkened image exposure environment, such as by placing the imagingarea in a dark box or by placing the entire system in a dark room.

The exemplary embodiment reduces delay in processing time by using aflat-panel imager to pre-screen the cell samples, which is much fastercompared to current methods. The flat-panel imager can image the entirearea of the substrate simultaneously and in parallel. Because the imagerdirectly converts images into digital form, processing time for film iseliminated.

Other advantages and benefits of the present exemplary embodiment willbecome apparent to those of ordinary skill in the art upon reading andunderstanding the following detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The exemplary embodiment may take form in various components andarrangements of components, and in various steps and arrangements ofsteps. The drawings are only for purposes of illustrating variousaspects and are not to be construed as limiting the exemplaryembodiment.

FIG. 1 is a block diagram of a method used to detect rare cellsimplemented in accordance with the exemplary embodiment.

FIG. 2 is a schematic illustration of a system in accordance with theexemplary embodiment.

FIG. 3 is a schematic of another system in accordance with the exemplaryembodiment.

FIG. 4 is a graph showing the number of electrons detected versus thetotal integration time for three different frame read outs in anexemplary embodiment system.

FIG. 5 is a graph showing the signal-to-noise ratio versus totalintegration time for three different frame read outs in an exemplaryembodiment system.

DETAILED DESCRIPTION

The exemplary embodiment systems and methods are particularly directedto detecting the presence of tagged biological agents in a sample. Thesystems and methods are adapted for qualitatively and quantitativelydetecting cells, and specifically, rare cells. Although the exemplaryembodiment systems and methods are described in terms of rare celldetection, it will be understood that the exemplary embodimentencompasses other systems and methods for detecting, reviewing,analyzing, or sampling biological agents.

The current art in digital imaging systems includes amorphous siliconarrays and large area charge-coupled diode (CCD) devices. Key factors inthe performance of such devices include the pixel pitch and the fillfactor. In the exemplary embodiment described herein, the pixel pitchrefers to the length of one side of a pixel, which is generally square.The pixel pitch determines the number of photons that can be detectedand traced to a specific spatial location. The fill factor refers to thepercentage of each pixel that can be used to detect the photons. Ahigher fill factor translates into increased sensitivity because fewerphotons fall into the “gaps” in and between pixels where they cannot bedetected. A higher fill factor also aids in noise reduction and imageresolution by increasing the detected signal. Amorphous silicon arrayscan be produced in large sizes, have a pixel pitch of about 100 to 127μm, and are relatively inexpensive. Large area CCDs have superior imagequality because they can have very fine pixel size and a high fillfactor, but they are difficult to produce in large sizes andconsequently have a high cost. “Linear” CCD arrays are also used inimaging, such as in scanners and copiers, but have moving parts forscanning an area. Moving parts reduce the life expectancy of the deviceand also increase the difficulty of manufacture.

In one aspect, the exemplary embodiment system uses a large areaflat-panel imager to detect and spatially locate rare cells through CL.The term “large area” with reference to a flat panel imager as describedherein refers to imagers having an imaging area or an image formingsurface of at least about 100 cm², such as provided by an imaging areaof 10 cm by 10 cm. Typically, this refers to imagers having an imagingarea of greater than 200 cm², greater than 400 cm², greater than 600cm², greater than 800 cm², greater than 1000 cm², and approximately 1200cm². The exemplary embodiment includes the use of imagers having animaging area greater than 1200 cm². A flat-panel imager is an imagerwhich uses a detection means, such as photoconductors, in the form of aflat surface. Currently, such imagers are available from companies suchas General Electric and Dpix for use in medical imaging, non-destructivetesting, security imaging, and research. Several types of imagers arediscussed in U.S. Pat. No. 6,272,207, the content of which isincorporated by reference herein. The exemplary embodiment can utilize aflat panel imager that includes a two dimensional array of photosensingdevices. In particular, an imager can be comprised of thin filmtransistors (TFTs) on an amorphous silicon substrate. Rahn et al.discusses these TFTs in Proc. SPIE, 3770: 136-145 (1999), the content ofwhich is incorporated by reference herein. TFTs have been demonstratedon a single substrate of dimensions of approximately 30 cm by 40 cm witha pixel pitch of 100 to 127 μm. This pixel pitch size gives good imageresolution and allows for a large dynamic range of detection.

A TFT array used in the exemplary embodiment compares well to a CCDarray. Atypical cell density when spread upon a substrate is 300,000 to500,000 cells per cm². With 10 μm diameter cell size, 50 million cellsprepared on a 100 cm² substrate will cover 39% of the surface, assuminga non-overlapping monolayer and no loss of cells during preparation.Actual coverage is likely less as cells may overlap and cell loss isexpected. On a flat-panel imager with square pixels with dimensions of100 μm, the 100 cm² substrate could be imaged with one million pixels.As target cells are ultra rare, more than one target cell in a pixel(100 μm by 100 μm) might occur, but only at very low frequency. As manyas 100 “potential hits” might occur, which could include 50 truepositives and 50 false positives. These 100 “potential hits” are furtherinspected with a high-magnification imaging device (such as a microscopeand a sensitive high quantum efficiency CCD camera) to obtainhigh-resolution images for confirmation. For example, through a 40×objective, a back-thinned back-illuminated CCD camera (e.g. HamamatsuORCA II BT 512) having 512 by 512 square pixels with effective pixelsize of 12.29 μm can image an area of about 157 μm by 157 μm. Withinthis image high resolution of a potential positive rare cell and manynon-rare cells would provide a good context for positive identificationof a true rare cell. The lower resolution of the flat-panel imager dueto its 100 μm pixel pitch would not affect the subsequent resolution ofthe microscope image which is used for confirmation. It is understoodthat CCD cameras with higher resolution and better sensitivity can beemployed if necessary. It may also be possible to use an imageintensifier coupled with a sensitive camera for detecting ultra lowlight in a much shorter time.

In another aspect of the exemplary embodiment, a method is provided fortagging target cells, cell fragments, and cell components such asproteins with a luminescent molecule. In one approach,immunocytostaining method is used to tag the target. In anotherapproach, a nucleic acids-based method similar in principle tofluorescent in situ hybridization (FISH) is used. Unlike immunostainingor FISH, where fluorochromes are conjugated to antibody or probing DNA,in CL detection enzymes capable of cleaving chemical bonds of CLreagents to generate lights are conjugated to the antibody or DNA. CLreagents are then applied onto the sample to allow chemiluminescence tobe generated at targeted sites. In yet another approach, radioisotopesare used in place of the CL enzyme to tag the target. Multiple taggingmethods (including fluorescence) could also be used simultaneously onone cell sample to more precisely locate target cells. Multiple probescontaining different enzymes that react with different CL reagents togenerate light of different wavelengths can also be used to target oneor more cellular markers. In this case, proper wavelength-specificfilters are implemented on the imager to separate these probes.

In accordance with the present exemplary embodiment, a method isprovided for qualitatively and quantitatively detecting cells. Cells arespread across a substrate (e.g., a slide). The cells are tagged withantibodies that carry an enzyme that will cause luminescence whensuitable reagents are applied. The substrate-sample is then placed on aflat-panel imager that detects the luminescence and pre-screens thecells. Digital processing is used to eliminate some false positives andto provide the spatial data necessary for a high-resolution imagingdevice, such as a high-power microscope to reexamine the cells in thatarea for confirmation of the diagnosis. A recording device, such as aCCD camera or a complementary metal oxide semiconductor (CMOS) sensorwith associated digital storage medium, also records the high-resolutionimage for later viewing and/or analysis. Confirmation of the diagnosiscan occur through human intervention, automatic identification, or othertechniques.

An exemplary embodiment method for detecting rare cells using aflat-panel imager and tagging is given in FIG. 1. A cell sample isprovided 110 which contains the desired target rare cells. The cellsample may come from any natural source and be of any type of cell, butthe cell sample is typically in the form of a blood sample containingblood components such as plasma, red blood cells, white blood cells, andthe like. The cell sample is then processed. Some of the components ofblood, e.g., red blood cells, may be removed. If the tagging methodrequires access to cell components inside the cell, then the cells inthe cell sample would need to be fragmented, broken up or permeabilizedto allow that access. Other processing may occur which enhances theimaging of the cell sample. The cell sample is then attached to asubstrate. Usually, the cell sample is spread as evenly across thesubstrate as possible. Cells will adhere to the substrate or theadhesion may be promoted with certain “conditionings” on the substrate.These substrate surface conditionings may include a layer or layers ofamino acids, collagen, amino silane, etc., just to name a few; or itcould be a nylon or nitrocellulose coating to enhance thechemiluminescent signals. The cells and/or cellular components areusually fixed with a fixing agent to allow for handling, preservation,and other reasons. The substrate is usually a slide; however, the slidemay be customized to enhance certain properties or have a form whichaids in diagnostic processing. Larger substrates (e.g., having the sizeof a standard wellplate or larger) that can hold one or more patientsamples are particularly suitable for this detection method. Slides orother sample receiving surfaces can include one or more coatings thatfacilitate binding of cells thereto. Generally, such coatings arecompatible with CL detection. Tagging agents or probes, e.g. antibodyconjugated with CL enzyme, are added to the cell sample 120. After thesetagging agents have attached to the target rare cells, several washingsteps may be included to reduce or eliminate non-specific binding. CLreagents are then added and spread evenly. The substrate is then placedon the flat-panel imager for imaging 130. The cell sample is analyzed atlow resolution 140 and “potential hits” are identified 150. Thisanalysis and identification is usually performed by electronic meanssuch as a computer, but may also be performed using human intervention.The coordinates of these potential hits are recorded relative to certainprescribed calibration marks on the substrate and then these coordinatesare translated into stage movements. Next, a high-resolution imagingdevice is used to reexamine the cells identified as “potential hits” 160and confirm the diagnosis. This can be done by either moving the stageholding the substrate-imager assembly or by moving the microscope andCCD camera. In this way, detection of tagged rare cells and rejection offalse positives are made possible via inspection of the high-resolutionimages. Again, this confirmation may be performed by electronic means orby human intervention.

An exemplary method for preparing cells to be analyzed on a flat-panelimager, e.g. steps 110-130 depicted in FIG. 1, is as follows:

1) A conventional or custom (large) microscopy slide (substrate) isprovided which has a defined “well structure” which has its chemistryoptimized for mammalian cell adhesion (such as the Erie CLEARCELL orADCELL chemistry) inside the well and which is surrounded by ahydrophobic chemistry or coating that will repel the aqueous solutionused for cell deposition.

2) Blood samples of patients are processed with NH₄Cl buffer to lyse redblood cells. The samples are then centrifuged, washed with phosphatebuffered saline (PBS) to remove erythrocytes and NH₄Cl, and re-suspendedin PBS. An appropriate amount of sample is pipet-transferred onto thesubstrate. The solution is spread evenly across the substrate with ablade to form a thin layer. Incubation occurs at 37° C. with highhumidity for 40 min followed by an addition of growth medium and furtherincubation of 20 min to promote cell adhesion.

3) Cells are then fixed by 2% paraformaldehyde for 20 minutes at roomtemperature followed by 2 rinses for 3 minutes each in PBS.

4) Non-specific binding is blocked by incubation with 20% human AB serumfor 20 minutes at 37° C.

5) After tapping off the serum, an appropriate amount of primaryantibody diluted in the same serum is added and the substrate isincubated for an hour. An example would be 1:100 dilution for mouseanti-human cytokeratine antibody (Monoclonal anti-Pan Cytokeratin, Sigmacatalog #C-2562). After one hour, the antibody solution is rinsed offwith PBS.

6) An enzyme-linked secondary antibody (e.g. alkaline phosphatase linkedgoat anti-mouse IgG) is diluted in AB serum and an appropriate amount isapplied into the well. The substrate is incubated for 30 minutes and theantibody solution is then rinsed off twice with PBS.

7) An appropriate amount of chemiluminescent reagent (such as CDP-Star,CSPD, and perhaps also enhancer if necessary) is added to the well at aconcentration specified by the manufacturer and the slide is coveredwith a cover slip and sealed with glue.

8) The substrate is assembled on the flat-panel imager for exposure.

An exemplary embodiment system 200 is shown in FIG. 2. A conventionalmicroscopic slide or slide assembly 250 on which tagged cells 240 areattached is aligned with a flat-panel imager 260 with the sample sidefacing the imager. If desired, a removable mirror 230 can be disposed ontop of the slide or slide assembly to reflect the photons emitted awayfrom the imager back towards the imager to increase the efficiency ofdetection. The pixel coordinates of the “potential hits” identified bythe flat-panel imager are recorded and translated into spatial data thatcan be subsequently used by a microscope 220 and camera 210 withcomputer-controlled movement 270 to accurately image those pixels inhigh resolution. While it is envisioned that the microscope and camerawill be moved during the high-resolution imaging phase, in analternative embodiment the microscope and camera remain fixed in placeand the microscope stage is moved instead; or both of them can movesimultaneously. It is also envisioned that the microscope and camera aremoved automatically, but in an alternative embodiment a human operatorcan move and/or control the microscope and camera.

FIG. 3 shows another exemplary embodiment system 300. Here, an imager390 is configured in the form of a wellplate. A slide 380 on whichtagged cells 370 are attached or otherwise deposited is disposed on theimager 390 with the assistance of an alignment system 360. Because ofthe alignment system 360, there is no need for optical alignment. Inthis exemplary embodiment, the alignment system 360 is depicted as fourcorner posts which define how the slide should be positioned withrespect to the imager 390. However, the alignment system can take otherforms, either mechanical forms where the slide is positioned to a knownposition with regards to the imager, or electronic forms where acomputer can be used to translate a relative position into an absolutecoordinate system. The imager/slide assembly is placed on a wellplateholder on a stage 395 of a microscope. While the microscope stage andflat-panel imager are described and depicted here as two separatesystems, in an alternative embodiment the imager serves as themicroscope stage and they are an integrated one-piece assembly.Immediately after the potential hits are logged by the imager, highresolution images are acquired by a 40× objective microscope 350 and asensitive CCD camera 340 with low noise that allows long exposure ifnecessary. 40× is only an example, as the exemplary embodiment includesthe use of microscopes having a objective magnification of from about10× to about 100×, and particularly about 20× to about 63×. In caseswhere imager and CCD operate simultaneously, a long working distanceobjective can be used which allow exposure through the transparentsubstrate. Detection processing equipment 330 directly translates the“potential hit” coordinates identified by the imager into data thatcause stage positioning equipment 320 to move the stage so the“potential hit” is located under the high-resolution microscope.Microscope focusing equipment 310 can move the stage in Z-direction sothe “potential hit” is in the focal plane of the high-resolutionmicroscope. Generally, the resolution of the camera is greater than theresolution of the imager, however the exemplary embodiment includesalternate configurations.

In both of the systems 200 and 300, depicted in FIGS. 2 and 3, theoperation and/or processing is generally performed in a darkenedenvironment. This can be accomplished by the use of a dark box orplacing the system in a dark room. The reason for this is that adarkened environment excludes or at least significantly reduces anyexternal photons.

In another exemplary embodiment, the high-resolution imaging device canbegin acquiring a high-resolution image while the flat-panel imagercontinues to identify “potential hits.”

In yet another exemplary embodiment, multiple substrates can be placedsimultaneously on the flat-panel imager to be imaged in parallel. It isenvisioned that 100-200 cm² is required to image a typical patient'ssample. At currently demonstrated substrate dimensions for TFTs of 30 cmby 40 cm, 12 substrates for 6-12 patients could be simultaneouslyprocessed. These numbers are exemplary and should not be construed aslimiting.

In other exemplary embodiments, optional devices are provided to allowfor different forms of imaging or for chemical reactions to beperformed. For example, pipettes could be utilized. The slide sampleswould first be inserted into the imager and an image of the backgroundlight would be taken. Then reagents would be added through the pipettesto induce CL. The background light could be subtracted from the CL lightsignal to increase sensitivity. Other equipment could be provided forother forms of microscopy to be performed. For example, visible whitelight microscopy, phase contrast microscopy, DIC microscopy, andfluorescent microscopy require a light source and/or filters but couldprovide additional information such as additional markers or morphologyregarding the targeted rare cells. In this case, cell sample may belabeled with CL probes as well as fluorescent probes. In certainversions, a system in accordance with the exemplary embodiment cancomprise one or more filters in which the filters substantially passlight within a particular range of wave lengths and/or substantiallyblock light outside that range of wave lengths. In addition to orinstead of the use of the noted filters, an exemplary embodiment systemcan comprise one or more light sources in which the light sources emitlight within a particular range of wave lengths. Thus, the exemplaryembodiment system and methods can detect the monochrome emission oflight from tagged samples, or detect multiple colors emitted from taggedsamples. One or more filters can be used to selectively absorb or blockcertain wavelengths of light. Multiple optical filters can be usedconcurrently to form stacks of filters as known by those skilled in theart. The large area flat panel imagers can be used to detect multiplecolors emitted from one or more tagged samples.

Calculations supporting the use of flat-panel imagers with CL areprovided herein. Previous experiments using CL detection for HeLa cellscontaining 10-50 copies of the target DNA reported a mean value of 115emitted photons/sec/cell and a mean value of 844 emittedphotons/sec/cell for CaSki cells containing 400-600 copies of the targetDNA. See Musiani et al. In immunocytostaining for rare cells, cellularprotein is usually the target instead of DNA. Depending on theparticular target protein marker, the number of target proteins per cellvaries. Fetal or cancerous cells often contain more than 100 copies ofmembrane or cytosolic proteins while chimerical cancerous genomic DNAstretches also exist at higher than 100 copies per cell. Calculationsassume a value of 1.8 emitted photons/sec/tagged protein and 10 copiesof the protein with each cell.

The quantum efficiency of a typical flat-panel imager is approximately75% at 600 nm. The wavelength of the maximum quantum efficiency can beengineered to match the wavelength of emitted photon from CL reagent,which is typically between 400 and 700 nm for most commonly used CLreagents such as Luminol, CSPD or CDP-Star.

The noise performance of a typical, commercially available flat-panelimager (with detector area 20 cm by 24 cm and 127 μm by 127 μm squarepixel) can be expressed as:

σ_(total)=√{square root over (σ_(ADC) ²+σ_(line) ²+σ_(pixel)²)}=√{square root over ((1069500)+(4.5×10⁻¹⁵ ×t)/1.6×10⁻¹⁹)}{square rootover ((1069500)+(4.5×10⁻¹⁵ ×t)/1.6×10⁻¹⁹)}(e _(RMS))

where t is the integration time for each frame of digital image. Thetotal noise of the flat-panel imager is dominated by electronic read outnoise for short frame times. For long integration times, the noiseincreases as a function of the square root of integration time becauseof the shot noise component of the detector dark current.

The integrated signal as a function of integration time can be estimatedas S=0.75×1.8×10×t, where 0.75 is the quantum efficiency, 1.8 is thefluence of the CL reagent, and 10 is the number of tags attached to thetarget cell. FIG. 4 shows the comparison of the integrated signal andnoise versus integration time. It clearly shows that integrated signalis larger than the noise for integration time t>180 seconds (3 min). Toachieve S/N=3, however, requires t>1426 seconds. For flat-panel imagers,constant dark current will saturate the photosensor signal if the frametime is set to such a relatively long period. The solution to thissaturation problem is to break the long integration time into n read outframes with frame time t/n and to add the n read out images digitally.The noise power of individual frames is added together such that thetotal noise can be expressed as:

σ_(total)=√{square root over ([(1069500)+[(4.5×10⁻¹⁵ ×t)/(1.6×10⁻¹⁹×n)]]×n)}{square root over ([(1069500)+[(4.5×10⁻¹⁵ ×t)/(1.6×10⁻¹⁹×n)]]×n)}{square root over ([(1069500)+[(4.5×10⁻¹⁵ ×t)/(1.6×10⁻¹⁹×n)]]×n)}

where t is the total integration time and n is the number of read outsduring the entire exposure process. The curves in FIG. 4 show the totalnoise for 1, 10, and 60 read out frames respectively and FIG. 5 showsthe signal-to-noise ratio versus time for 1, 10, and 60 read out frames.For 10 read out frames, it is possible to achieve S/N>3 at 30 min, or aframe time of 180 seconds. For 1.8 fA dark current and 0.7 pF sensorcapacitance, the constant dark current will generate 0.46 V voltageshift on the sensor electrodes, which is well within the dynamic rangeof typical PIN photo sensors.

At actual frequencies, the number of photons emitted is 10-fold higherthan used for the calculation above, resulting in 10-fold shorterintegration times, or 3 min only. The number of signaling molecules canalso be amplified with an additional layer of antibody during samplepreparation without proportionally increasing the noise. This would alsoreduce the integration time required for imaging. Direct X-ray photondetection has been demonstrated to show the capability of single-photondetection. Therefore, a high sensitivity can also be expected forradioisotope tags. Flat-panel imagers with pixel level pre-amplifiershave also been successfully demonstrated.

The exemplary embodiment has been described with reference to thepreferred embodiments. Obviously, modifications and alterations willoccur to others upon reading and understanding the preceding detaileddescription. It is intended that the exemplary embodiment be construedas including all such modifications and alterations insofar as they comewithin the scope of the appended claims or the equivalents thereof.

1. A method for detecting the presence of tagged biological agents in asample, the method comprising: providing a system having an imager, amicroscope, a camera, and a sample receiving surface; providing a samplecontaining a biological agent to be detected; depositing the sample uponthe sample receiving surface of the system; tagging the biological agentto be detected within the sample; obtaining a digital image of thedeposited tagged sample from the system; analyzing the digital image toselect one or more regions of the tagged sample for high-resolutionexamination; positioning the imager, microscope, and camera relative toeach other so the microscope can image a selected region of the taggedsample determined in the analyzing step; obtaining a second digitalimage of the selected region of the tagged sample; and confirming thedetection of the tagged biological agent.
 2. The method of claim 1,wherein the step of tagging is performed by tagging the biological agentusing an antibody-based method or a DNA-based method withlinked-enzyme(s) for chemiluminescent detection and/or withfluorochrome(s) or radioisotope(s).
 3. The method of claim 1, whereinmultiple tagging methods are performed on the same sample.
 4. The methodof claim 1, wherein the step of depositing is performed by depositingthe sample on a substrate and then placing the substrate on the samplereceiving surface of the system.
 5. The method of claim 1, wherein thestep of obtaining the first digital image from the system is performedby detecting a range of wavelengths emitted by the tagged sample.
 6. Themethod of claim 1, wherein the step of analyzing is performed bydetermining the regions of the sample which emitted light at a range ofwavelengths above a threshold, the threshold being determined by humanor electronic means.
 7. The method of claim 1 wherein the step ofpositioning is performed using electronic positioning equipment to moveone or more of the imager, the microscope, and the camera.
 8. The methodof claim 1, wherein the step of obtaining the second digital image isperformed by using the microscope in conjunction with the camera tocapture the second digital image.
 9. The method of claim 1 wherein thesecond digital image has a higher resolution than the first digitalimage.
 10. A method for detecting the presence of tagged biologicalagents in a sample by use of a system having, (i) a large area imagerhaving an image forming surface, the imager being able to form digitalor electronic images of the sample free from intermediate image forminglenses, (i)) a selectively positionable microscope and camera in viewingrelation with the image forming surface, the camera adapted to acquirean image through the microscope in the form of digital data, and (iii)an electronic controller in communication with the imager, microscope,and camera, and configured to position at least one of the imager,microscope, and camera relative to each other in order to image selectedregions of the image forming surface, the method comprising: providing asample containing a biological agent to be detected; depositing thesample upon the sample receiving surface of the system; tagging thebiological agent to be detected within the sample; obtaining a digitalimage of the deposited tagged sample from the system; analyzing thedigital image to select one or more regions of the tagged sample forhigh-resolution examination; positioning the imager, microscope, andcamera relative to each other so the microscope can image a selectedregion of the tagged sample determined in the analyzing step; obtaininga second digital image of the selected region of the tagged sample; andconfirming the detection of the tagged biological agent.
 11. The systemof claim 10, wherein the microscope contains an objective lens ofmagnification power of from about 10 to about
 100. 12. The method ofclaim 10, wherein the system further comprises one or more filters, thefilters substantially passing light within a range of wavelengths andsubstantially blocking light outside the range of wavelengths.
 13. Themethod of claim 10 wherein the sample receiving surface includes acoating that facilitates binding of cells thereto.
 14. The method ofclaim 13 wherein the coating is compatible with chemiluminescentdetection.
 15. The method of claim 10, further including matching awavelength of a maximum quantum efficiency of the large area imager to awavelength of an emitted photon from a chemiluminescent reagent in thesample.
 16. The method of claim 10, further including designing aquantum efficiency of the large area imager to match a wavelength ofphotons emitted from a selected reagent in the sample which are emittedat a rate of between about 400 to about 700 nm.
 17. The method of claim10, further including adding noise of individual frames together suchthat the total noise can be expressed by a calculation:σ_(total)=√{square root over ([(1069500)+[(4.5×10⁻¹⁵ ×t)/(1.6×10⁻¹⁹×n)]]×n)}{square root over ([(1069500)+[(4.5×10⁻¹⁵ ×t)/(1.6×10⁻¹⁹×n)]]×n)}{square root over ([(1069500)+[(4.5×10⁻¹⁵ ×t)/(1.6×10⁻¹⁹×n)]]×n)} where t is the total integration time and n is the number ofreadouts during the entire exposure process
 18. The method of claim 17,wherein as a frequency of the system increases, a number of photonsemitted increases compared to the photons used in the calculation,resulting in shorter integration times.
 19. The method of claim 18,wherein the number of photons emitted is tenfold higher than used in thecalculation, resulting in tenfold shorter integration times.
 20. Amethod for detecting the presence of tagged biological agents in asample, the method comprising: forming on a large area imager having animage forming surface, digital or electronic images of the sample freefrom intermediate image forming lenses; positioning a selectivelypositionable microscope and camera in viewing relation with the imageforming surface; acquiring by the camera an image through the microscopein the form of digital data; positioning at least one of the imager,microscope, and camera relative to each other in order to image selectedregions of the image forming surface and integrate a time period (t),and a number (n) of read-out frames with a frame time (t/n); and formingimages by integrating the images over the integrating time period (t),including breaking the image integrating time period (t) into the number(n) of read-out frames to avoid noise saturation of the images.